1. Field of the Invention
The present invention relates to a recombinant mammal cell and a method of producing a protein with use of the cell.
2. Background Art
There are known a variety of recombinant protein producing systems with procaryotes or eucaryotes as a host cell. According to the recombinant protein producing system with a mammal cell as the host cell, it is possible to subject proteins derived from higher animals including human to post-translational modifications such as the addition of polysaccharide chain, folding and phosphorylation in a similar manner to those produced in vivo.
The post-translational modification is necessary for reproducing physiological activities of a native protein in the recombinant protein. Thus, a protein producing system with a mammal cell as the host cell is preferably used in recombinant protein producing systems used in medicaments for which such physiological activities are particularly needed.
In the industrial production of pharmaceutical proteins, it is important to stably maintain the expression of a protein in a high level. Particularly, the maintenance of the stable expression level is important not only to the respect of cost but also to the verification of the identity and safety as pharmaceutical proteins. In order to use the recombinant protein producing cell for its production in industrial scales, it is necessary to magnify a scale for culturing the clone of the recombinant protein producing cell. It is usually estimated for magnifying the scale that the clone just developed must be subjected to at least 60 cell divisions (Brown, M. E. et al. (1992) Cytotechnology, 9, 231-236.), and the expression level must be maintained constant during the cell division.
The specific productivity of the recombinant clonal cell may also be reduced to such a level that is hardly used as production cells during or after period for magnifying the culture scale, and in this case the development period over several months will come to nothing (Barnes, L. M. et al. (2003) Biotechnol. Bioeng. 81, 631-639).
The use of selection drugs during the periods of scale-up and practical production rises the costs not only of culture but also of purification processes in order to avoid the risk of polluting pharmaceuticals with toxic agents. Thus, the production of the recombinant clonal cell is generally carried out without addition of selection drugs.
The recombinant mammal clonal cell which produces the objective protein is required to have the specific productivity at high level as well as the property which enables to stably maintain the specific productivity level without addition of the selection drug.
Specific productivity in high levels is generally achieved by a process for increasing the copy number of an exogenous gene which codes for an objective protein by gene amplification technology, and systems such as CHO-DHFR and GS-NSO have been established as practical techniques (Japanese Patent Publication No. 7-40933; Werner R. G. et al. (1998) Arzneim.-Forsch./Drug Res 48, 870-880, and the like).
However, it has been confirmed that when clonal cells having increased levels of specific productivity are selected and then the selected clonal cells are continuously cultured in a medium containing no selection drugs, the level of specific productivity is lowered or vanished in most of the clones. Furthermore, it has also been described in literatures that the expression level of an objective protein is not always increased in proportion to the increased copy number of an integrated gene (Japanese Patent Application Laid-Open Disclosure No. 2002-541854; Kim, N. S. et al. (1998) Biotechnol. Bioeng., 60, 679-688; and the like).
It has been reported that the decreased level of specific productivity or the disappeared production of the objective protein are primarily caused by the decreased copy number of genes (Kim, N. S. et al. (1998) Biotechnol. Bioeng., 60, 679-688, Kim, S. J. (1998) Biotechnol. Bioeng. 58, 73-84, Yoshikawa, T. et al. (2000) Biotechnol. Progr. 16, 710-715; and the like).
It has been described in the literatures as Repeat-Induced Gene Silencing (RIGS) that such decrease or disappearance of the specific productivity levels are also caused by repeated integrations of multiple gene copies of identical sequence in tandem (Henikoff, S. (1998) Bioessays 20, 532-535; Garrick, D. et al. (1998) Nat. Genet. 18, 56-59). It has been reported that RIGS may be caused also in 2-3 copies of gene (McBurney, M. W. (2002) Exp. Cell Res. 274, 1-8).
Furthermore, no satisfactory solutions have hitherto been presented with respect to stability of the specific productivity level of an objective protein. Clonal selection is now empirically carried out on the basis of data which have been accumulated with regard to the growth rate and productivity during the culture of clone for a long period. According to this empirical process, it is rarely accomplished to get a clonal cell having a stable specific productivity level of the objective protein, which can probably be obtained by chance (Barnes, L. M. et al. (2003) Biotechnol. Bioeng. 81, 631-639).
The present inventors have previously reported a method for obtaining a recombinant cell in which an expression unit of the green fluorescent protein (GFP) as a single copy is integrated into the locus of the hypoxanthine-phosphoribosyl transferase (hprt) gene by homologous recombination (Biotechnol. Bioeng., 95(6): 1052-1060, 2006). The clonal cell stably maintains the specific productivity level even during its cultivation for a long period in the absence of the selection drug.
In the case of obtaining usual random recombinant clonal cells, multiple copies of exogenous genes are often integrated into the identical chromosomal site (Martin, D. I. K. and Whitelaw, E. (1996) Bioessays, 18, 919-923). These copies may be a target of RIGS. On the contrary, it is possible to avoid RIGS in the integration of one copy by homologous recombination (Whitelaw, E. et al. (2001) Methods in Mol. Biol. 158, 351-368).
On the other hand, it is known also in the integration of one copy that the stability of the specific productivity level largely varies depending on chromosomal sites for integration (Walters, M. C. et al. (2007) Genes Dev. 10, 185-195).
It can be found from these facts that the hprt genetic site having a copy of an exogenous gene integrated therein is excellent for maintaining the stability of the specific productivity level. On the other hand, it is however impossible to estimate the stability of the specific productivity level as well as the possibility of avoiding RIGS when multiple copies of an exogenous gene are integrated into the hprt genetic site.
In addition, Japanese Patent Application Laid-Open Disclosure No. H7-500969 discloses that an erythropoietin gene expression unit has been integrated into the hprt gene locus of HT1080 cell derived from human fibrosarcoma by homologous recombination. However, no expression of erythropoietin gene has been confirmed, nor integration of multiple copies has been found.